Part:BBa_K3020000:Experience

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Typical DNA damage agents include nalidixic acid (NA) (Sigma), formaldehyde (formaldehyde, CH2O) (Sigma) and hydrogen peroxide(H2O2).The following non-DNA damage reagent was used as a negative control:kanamycin sulfate，KAN）（Amresco）,acetone,phenol. The reagent concentration and gradient were calculated and added to the 40 ml bacterial solution (LB) system according to the following induction volume.

DNA damage agent concentration gradient and induced addition

non-DNA damage agent concentration gradient and induced addition

The BL21 strain introduced with Part:BBa_K3020000 was cultured overnight in LB medium (AMP resistance) at 37°C, and inoculated into fresh LB liquid medium (AMP resistance) at 1:50 inoculation, and shake cultured at 37 ° C for 3 hours. The OD600 is 0.2-0.4. Add DNA damaging agents and non-injury agents with different concentration gradients. The concentration and dosage are shown in the above figure. Induction at 37°C 130 r / min for 2-3 h. Standard error was taken three times for each set of experiments.

date analysis

OD600 represents the absorbance or optical density of a sample measured at a wavelength of 600 nm, which is a common method for estimating the concentration of bacteria or other cells in a liquid. The measured concentration can indicate the stage of culturing the cell population, ie whether it is in a lag phase, an exponential phase or a stationary phase.

Relative optical density ratio ROD=ODx/OD0

The formula is the relative optical density ratio calculation formula, OD x is the OD 600 value of the experimental group, OD0 is the OD 600 value of the control group without adding the damaging agent at the same time, and the ROD reflects the toxicity to the bacteria exposed to known or unknown compounds.

specific fluorescence units SFU=RFU/OD 600

The formula is the specific fluorescence intensity, where RFU is the relative fluorescence intensity unit measured by the microplate reader, OD 600 is the OD value of the same sample, representing the fluorescence intensity per unit OD, and RFU reflects the fluorescent protein expression intensity of the cells. This unit avoids deviations in the calculation of fluorescence intensity due to differences in the number of bacteria between different samples.

induction factor Fi=SFUx/SFU0

Where SFUx is a sample treated with a toxic compound and SFU0 is a control sample at the same time point. In this experiment, the inducing factor Fi value was used to evaluate the ability of the compound to damage the reagent. It can be seen from the literature that when the induction time is 2 h, the Fi factor value of any compound at any concentration reaches 2 or higher. This compound was identified as a DNA damaging agent.

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